Articular cartilage is composed of chondrocytes encased within the complex extracellular matrix produced by these cells. The unique biochemical composition of this matrix provides for the smooth, nearly frictionless motion of articulating surfaces of the knee joint. With age, tensile properties of human articular cartilage change as a result of biochemical changes. After the third decade of life, the tensile strength of articular cartilage decreases markedly. Damage of cartilage produced by trauma or disease, e.g., rheumatoid and osteoarthritis, can lead to serious physical debilitation.
The inability of cartilage to repair itself has led to the development of several surgical strategies to alleviate clinical symptoms associated with cartilage damage. More than 500,000 arthroplastic procedures and joint replacements are performed annually in the United States alone. Autologous chondrocyte implantation is a procedure that has been approved for treatment of articular cartilage defects. The procedure involves harvesting a piece of cartilage from a non-weight bearing part of the femoral condyle and propagating the isolated chondrocytes ex vivo for subsequent implantation back into the same patient (Brittberg et al. (1994) New England J. of Medicine, 331: 889–895).
Articular chondrocytes express articular cartilage-specific extracellular matrix components. Once articular chondrocytes are harvested and separated from the tissue by enzymatic digestion, they can be cultured in monolayers for proliferative expansion. However, during tissue culture, these cells become phenotypically unstable, adopt a fibroblastic morphology, and then cease to produce type II collagen and proteoglycans characteristic of hyaline-like articular cartilage. Such “dedifferentiated” cells proliferate rapidly and produce type I collagen, which is characteristic of fibrous tissue. Nevertheless, when placed in an appropriate environment such as suspension culture medium in vitro (Aulthouse et al. (1989) In Vitro Cell. & Devel. Biology, 25: 659–668) or in the environment of a cartilage defect in vivo (Shortkroff et al. (1996) Biomaterials, 17: 147–154), the cells redifferentiate, i.e., express articular cartilage-specific matrix molecules again. The reversibility of dedifferentiation is key to the successful repair of articular cartilage using cultured autologous chondrocytes.
Human chondrocytes are typically cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) (Aulthouse et al. (1989) In Vitro Cell. & Devel. Biology, 25: 659–668; Bonaventure et al. (1994) Exp. Cell Res., 212: 97–104). However, even though serum is widely used for mammalian cell culture, there are several problems associated with its use (Freshney (1994) Serum-free media. In Culture of Animal Cells, John Wiley & Sons, New York, 91–99): 1) serum contains many unidentified or non-quantified components and therefore is not “defined;” 2) the composition of serum varies from lot to lot, making standardization difficult for experimentation or other uses of cell culture; 3) many of the serum components affect cell attachment, proliferation, and differentiation making it difficult to control these parameters; 4) some components of serum are inhibitory to the proliferation of specific cell types and to some degree may counteract its proliferative effect, resulting in sub-optimal growth; and 5) serum may contain viruses and other pathogens which may affect the outcome of experiments or provide a potential health hazard if the cultured cells are intended for implantation in humans.
Thus, the use of defined serum-free media is particularly advantageous in the ex vivo expansion of chondrocytes for treatment of cartilage defects. However, such defined serum-free media must be sufficient for attachment of adult human articular chondrocytes seeded at low density, sustain proliferation until confluent cultures are attained, and maintain the capacity of chondrocytes to re-express the articular cartilage phenotype.
There has been some effort to develop biochemically defined media (DM) for cell culture. DM generally includes nutrients, growth factors, hormones, attachment factors, and lipids. The precise composition must be tailored for the specific cell type for which the medium is designed. Successful growth of some cell types, including fibroblasts, keratinocytes, and epithelial cells has been achieved in various DM (reviewed by Freshney, 1994). However, attachment and proliferation of cells in the known media are often not optimal.
Additionally, the amounts of starting cell material available for autologous chondrocyte implantation are generally limited. Therefore, it is desirable to seed articular chondrocytes at a minimal subconfluent density. Attempts to culture articular chondrocytes at subconfluent densities in DM have been only partially successful. Although DM that can sustain the proliferative capacity of the chondrocytes seeded at low density have been developed, the use of these media still requires serum for the initial attachment of cells to the tissue culture vessel after seeding (Adolphe et al. (1984) Exp. Cell Res., 155: 527–536, and U.S. Pat. No. 6,150,163).
A need exists to optimize, standardize, and control conditions for attachment, proliferation and maintenance of redifferentiation-capable chondrocytes for use in medical applications, especially, in humans.